This invention relates to sensing of diffusion of biomolecules. More particularly, the present invention relates to substrates, apparatus and methods for monitoring and detecting interactions between a ligand and a receptor.
The drug discovery process is a highly risky venture. The Pharmaceutical Research and Manufacturers of America estimate the average time to bring a drug to market is approximately 12 to 15 years at an average cost of approximately 500 million dollars. Among US pharmaceutical companies, more than 100 new therapeutic treatments have been added to the available medicines in the last two years. Among the many sequence of events applied in the pharmaceutical industry to realize commercially successful products, high-throughput screening (HTS) is believed to be an essential cornerstone of an effective drug discovery strategy. The HTS market in 1998 was estimated at 1.6 billion dollars.
High-throughput screening (HTS) refers to the initial activity in the pharmaceutical development process that systematically compares the binding of a target molecule or compound with each compound archived in a pharmacophore compound library. These libraries may contain millions of potential drugs acquired from a variety of sources, natural or otherwise, that are systematically screened for bioactivity against target molecules or compounds. The remarkable biological and biochemical advances of the last decade at the cellular and molecular levels have created numerous opportunities for discovery by uncovering an abundance of new receptors and enzymes that are mechanistically associated with disease pathologies. As these new targets emerge, a demand on analytic capacity to screen the targets against the large compound libraries for xe2x80x9chitsxe2x80x9d becomes a tremendous logistical effort.
The development of a technology applying direct binding assays (DBA) to high-throughput screening (HTS) could capture a significant share of the market for HTS. Surface-plasmon resonance (SPR) is a popular DBA technique in the pharmaceutical industry. SPR is but one of a large class of optical biosensors collectively referred to as evanescent wave detectors. This class includes film waveguide grating couplers, film prism waveguide couplers and long-period fiber waveguide couplers. The essential feature of all these techniques is that a standing xe2x80x9cevanescentxe2x80x9d wave is generated above the sensing surface by a wavelength""s distance from the surface (approximately 100-200 nm) that is sensitive to the local dielectric environment. By changing the local refractive index, the standing wave is altered, requiring either a new angle of incident light to set up the xe2x80x9cresonance conditionxe2x80x9d or inducing a phase shift of the reflected light. Since all proteins, independent of sequence, contribute the same refractive index per unit mass, this technique can serve as a mass detector. A linear correlation between resonance angle shift and surface protein concentration has been demonstrated, allowing real time detection of mass change without the need for labeling. All evanescent wave techniques are variations on this essential theme.
To distinguish between molecules floating in the bulk solution from those molecules attached to surface-bound target molecules, evanescent-based biosensors require that one flows buffer solution over the sensing surface after having flowed the buffer/analyte solution to infer the presence of surface bound analyte. Measurement of the adsorption in real time requires complex fluidic control of different flow conditions and solutions to infer the association rate constants from the observed binding rates. This is because evanescent-based methods do not distinguish between molecules floating in the evanescent standing wave and molecules anchored to the substrate also present in the evanescent field.
Another limitation of evanescent wave methods is that they do not readily lend themselves to miniaturization. In addition, light is invariably coupled into substrates with non-zero angles-of-incidence, often requiring complex schemes to measure extremely small shifts in reflected light or inducing small angular shifts in the incident light. Such limitations make massive deployment of similar sensing elements on small chips extremely problematic.
It would be desirable to provide substrates, apparatus and methods that do not encounter the drawbacks of evanescent-wave based methods. Many drugs are small molecules in the range between 200-1000 Dalton with binding affinities in the range between 10xe2x88x926 and 10xe2x88x9212 M. Therefore, it would be advantageous to provide methods and apparatus capable of characterizing small-drug and target-protein binding interactions. It would also be advantageous to provide HTS methods and apparatus that are capable of being xe2x80x9chomogeneous and label-freexe2x80x9d and provide the same information content as the more labor-intensive methods discussed above.
The invention relates to assay methods and apparatus for monitoring biological or chemical interactions by providing means to monitor and/or detect diffusion proximate a sensing area. According to the present invention, the interactions between and among chemicals, cells and biomolecules can be detected by monitoring the diffusion of a molecule or chemical proximate a sensing area. Such diffusion monitoring provides the ability to detect and measure interactions between ligands and receptors, such as a protein molecule or a cell.
According to one aspect of the invention, an apparatus is provided that measures rate of diffusion proximate a sensing area. According to another aspect of the invention, the rate of diffusion may be measured by several means. For example, the rate of diffusion proximate the sensing area may be measured by monitoring the concentration of a molecule proximate the sensing area. One example of such an apparatus may include a first area that may optionally contain a matrix material and receptor molecule or cell contained in the first area. The matrix material can be any suitable matrix for the receptor, such as a polymeric matrix material. According to this aspect of the invention, the apparatus preferably includes a second area adjacent the first area and a boundary area is disposed between the first and second areas. According to this aspect of the invention, preferably a ligand molecule is contained in the first area with the receptor and the matrix material. The second area preferably contains a solution such as a buffer solution.
According to another aspect of the invention, the ligand is smaller than the receptor. For example, the ligand molecule may be a drug molecule having a molecular weight less than 1000 Daltons, and the receptor has a molecular weight greater than 5 kiloDaltons. According to another aspect, the boundary area includes a membrane operative to allow ligand molecules to pass therethrough and to prevent passage of receptor molecules In another aspect of the invention, the receptor includes a protein molecule. It is understood that the invention is not limited to any particular ligands and receptors. As such, the receptors could include a wide variety of biomolecules including, but not limited to proteins, nucleic acids, and cells.
According to still another aspect of the invention, the means for detecting the diffusion of the molecules or cells includes an optical detector. A wide variety of suitable optical detection systems can be used in accordance with the present invention. For example, a light source and a light detector can be used. The light source may be, for example, an ultraviolet (UV) light source, and the light detector can be a charge-coupled light detector. According to this aspect, the light may be directed towards the second area, and the optical detector is positioned and operative to measure the change in light absorbed by the second area.
According to another embodiment of the invention, the means for detecting the rate of diffusion of the molecules or cells includes a diffraction device that includes three laterally-spaced openings, slits or slots. The laterally-spaced openings or slots may be provided on a suitable substrate. According to this aspect of the invention, the means for detecting the rate of diffusion of the molecules or cells involves monitoring the change in the far-field diffraction pattern generated by the diffraction device. According to a preferred aspect of the invention, the three laterally spaced openings includes a central opening containing a ligand and receptor in solution. According to this aspect, means are provided for detecting the change in the concentration of the ligand and the receptor contained in the central opening. The means for detecting the change in concentration may include a charge-coupled device (CCD) camera or other suitable device for monitoring the change in the diffraction pattern generated by the three laterally spaced openings as the ligand diffuses from the central opening.
According to another aspect of the invention, a method of analyzing biomolecular binding is provided, which involves detecting the diffusion of biomolecules proximate a slotted diffraction surface. The slotted diffraction surface may include three laterally spaced slots or openings, or any other arrangement capable of producing a far field diffraction pattern. According to one aspect of the invention, a central slot is provided and a ligand and receptor in solution are positioned proximate the central slot.
Another aspect of the invention involves monitoring the rate of diffusion of ligands from an upstream area towards a downstream area. Preferably, the upstream area includes an upstream compartment containing a mixture of a ligand and a receptor. The ligand and receptor are preferably contained in a matrix material, for example, a polymeric matrix. Diffusion of the ligand and receptor towards the downstream area can be measured by measuring the change in absorbance of light in the downstream compartment. As a greater amount of ligand and receptor diffuse towards the downstream area, the absorbance of light in the downstream area will increase. The absorbance characteristics can be utilized to detect and quantify the binding between a ligand and a receptor.
The invention provides a relatively simple and flexible method to detect chemical reactions, biomolecular reactions, particularly interactions between ligands and receptors, thus facilitating high throughput screening of biomolecules and drug candidates. Direct sensing of ligand-receptor binding can be measured without the need for introducing fluidics. In contrast, evanescent wave detectors typically require flow of buffer solution over the sensing element after having flowed the buffer/analyte solution to infer the presence of surface bound analyte. It is envisioned that the present invention can be combined with fluidics techniques. It will be understood, however, that analysis of binding between a wide variety of ligands and receptors can be accomplished without resort to fluidics techniques.
The embodiment of the invention involving monitoring the diffraction pattern also has the advantage that many 3-slit sensing elements on small substrates are possible because of the small lateral dimensions attainable by the zero angle-of-incident design. In addition, it is possible to obtain high-sensitivity (in the range of two orders of magnitude) by introducing a quarter wave trough or raised-surface on the central slit of the tri-slit device. Furthermore, sensitivity is not compromised by thermal expansion of materials used to construct the device under room temperature operational conditions.
Additional advantages of the invention will be set forth in the following detailed description. It is to be understood that both the foregoing general description and the following detailed description are exemplary and are intended to provide further explanation of the invention as claimed.